Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates

The SARS-CoV-2 main protease (M^pro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the M^pro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed M^pro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the M^pro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the M^pro of the original strain will be effective in all the variants. Thus, M^pro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.